A characteristic of aging skin is that skin cells, particularly fibroblasts, progressively lose their ability to proliferate. A reduction in the number of viable skin cells leads to reduced synthesis and secretion of collagen and other extracellular matrix proteins, resulting in thinning and wrinkling of the skin. Aging skin is further characterized by a decrease in flexibility and tensile strength. As a result, aging skin becomes more vulnerable to damage caused by external stress. Also, due to unfavorable changes in its texture, aging skin does not heal as well as young skin. Thus, restoration of a healthy structure to aging skin is important not only from a cosmetic point of view but also for health reasons.
Agents that enhance proliferation of epidermal and dermal cells in the skin, including fibroblasts and keratinocytes, are likely to be effective in restoring or maintaining the strength and thickness of aging skin. Fibroblast cells, located in the dermal layer, play important roles in mechanisms such as wound healing by, for example, producing components of the extracellular matrix like collagen and various cytokines, which, in turn, enhance the proliferation and migration of keratinocytes. Keratinocytes are located in the epidermal layer and form a barrier against the external environment.
There are many cosmetic products, some of which contain placental extracts, that can temporarily enhance the look and quality of skin. Such cosmetics and their ingredients have been discussed, for example, in two books [Begoun, P., “Don't go to the cosmetics counter without me.” Beginning Press (1996); Winter, R. “Consumer's dictionary of cosmetic ingredients.” Three Rivers Press (1994)]. Cosmetics are not assumed to change the structure of the skin (other than increasing hydration and inducing other small changes), and therefore do not require FDA approval. While certain light and UV sources have been proposed for skin care, and claims are occasionally made that these sources enhance proliferation of fibroblasts and epidermal cells, it is not clear if these effects are indeed produced by the presently available commercial cosmetic products.
Induction of relatively long-lasting changes in the skin requires agents and/or procedures that enhance the number of viable epidermal cells (keratinocytes) and dermal cells (particularly fibroblasts). This can be achieved by either enhancing the lifetime of these cells and/or stimulating their proliferation. Local treatment by known growth factors may be able to do that. However, most powerful growth factors are prohibitively expensive. Furthermore, virtually all growth factors stimulate the proliferation of either fibroblasts or keratinocytes, but not both.
Administration of placental alkaline phosphatase, one of the presently known four members of the alkaline phosphatase enzyme family [Millan, J. L. and Fishman, W. H., “Biology of human alkaline phosphatases with special reference to cancer,” Critical Reviews in Clinical Sciences, 32, 1-39 (1995)], has been reported to enhance both the proliferation [She, Q.-B., Mukherjee, J. J., Huang, J.-S., Crilly, K. S. and Kiss, Z., “Growth factor-like effects of placental alkaline phosphatase in human and mouse embryo fibroblasts,” FEBS Lett., 469, 163-167 (2000)] and survival [She, Q.-B., Mukherjee, J. J., Chung, T. and Kiss, Z., “Placental alkaline phosphatase, insulin, and adenine nucleotides or adenosine synergistically promote long-term survival of serum-starved mouse embryo and human fetus fibroblasts,” Cellular Signalling, 12, 659-665 (2000)] of mouse embryo fibroblasts as well as fibroblast-like cells derived from the lung of human fetus.
Placental alkaline phosphatase is a member of the alkaline phosphatase group of enzymes that hydrolyze phosphate-containing compounds at alkaline pH. Mature placental alkaline phosphatase is a dimer of two identical glycosylated subunits. One source of placental alkaline phosphatase is human placenta, which synthesizes this enzyme during pregnancy so that toward the end of third term the enzyme's level in the placenta tissue and maternal/fetal blood becomes very high. Therefore, it is very unlikely that human placental alkaline phosphatase exerts toxic or pathological effects in human tissues. Subunits of human placental alkaline phosphatase (“PALP”) have an approximate molecular weight of 66 kDa, as determined by gel electrophoresis.
A determination of an in vivo half-life for human PALP was reported in 1965 [Clubb, J. S., Neale, F. C. and Posen, S., “The behavior of infused human placental alkaline phosphatase in human subjects.” J. Lab. & Clin. Med. 66, 493-507 (1965)]. In human subjects, injected PALP is reported to remain remarkably stable in the circulation, with an estimated biological half-life of about 7 days. In the reported experiments, PALP was injected as a minor constituent in a mixture of PALP and albumin obtained by extraction, without further purification. The authors reported that PALP up to serum concentration of 975 “King-Armstrong” (KA) units appeared metabolically inert, and hypothesized that PALP performs no measurable physiological function in circulation.
At least two potential therapeutic uses for human PALP have been reported. U.S. patent application Ser. No. 10/317,916, filed Dec. 12, 2002 and entitled “Placental Alkaline Phosphatase to Control Diabetes,” and U.S. patent application Ser. No. 10/441,992, filed May 20, 2003 and entitled “Placental Alkaline Phosphatase to Control Diabetes,” each of which is hereby incorporated by reference in its entirety, report the use of human PALP to reduce or control plasma glucose level. U.S. patent application Ser. No. 09/873,654, filed Jun. 4, 2001 and entitled “Compositions and Methods for Stimulating Wound Healing and Fibroblast Proliferation,” which is hereby incorporated by reference in its entirety, reports the use of human PALP in combination with growth factors or serum factors for wound-healing compositions.
As mentioned above, it has recently been reported that administration of PALP can enhance both the proliferation and survival of mouse embryo fibroblasts as well as fibroblast-like cells derived from the lung of human fetus. Although mouse embryo fibroblasts and fibroblast-like cells from the fetus lung differ from human skin fibroblasts in many respects, those results indicated that PALP may be effective to enhance proliferation of human skin fibroblasts and even human keratinocytes. If so, PALP may be useful for enhancing proliferation of skin cells in vivo, and for promoting thicker and stronger skin.
In the past, many attempts were made to use various placenta extracts to improve the quality of skin (reviewed in Begoun, supra). However, all these extracts, like any tissue extract, presumably contained hundreds or even thousands of unknown components, including numerous proteins and hormones, many of which certainly exhibit uncharacterized biological activities. In fact, as reported recently [Angelucci, C., Lama, G., and Sica, G., “The growth of malignant and nonmalignant human cells is modulated by a human placental extract,” Anticancer Res., 19, 429-436 (1999)], human placental extract contains both inhibitors and activators of cell growth whose effects depend on the incubation conditions in a highly unpredictable manner.